Clonorchis sinensis infection amplifies hepatocellular carcinoma stemness, predicting unfavorable prognosis

Background Extensive evidence links Clonorchis sinensis (C. sinensis) to cholangiocarcinoma; however, its association with hepatocellular carcinoma (HCC) is less acknowledged, and the underlying mechanism remains unclear. This study was designed to investigate the association between C. sinensis infection and HCC and reveal the relationship between C. sinensis infection and cancer stemness. Methods A comprehensive analysis of 839 HCC patients categorized into C. sinensis (-) HCC and C. sinensis (+) HCC groups was conducted. Chi-square and Mann–Whitney U tests were used to assess the association between C. sinensis infection and clinical factors. Kaplan–Meier and Cox regression analyses were used to evaluate survival outcomes. Immunohistochemistry was used to determine CK19 and EpCAM expression in HCC specimens. Results Compared to C. sinensis (-) HCC patients, C. sinensis (+) HCC patients exhibited advanced Barcelona Clinic Liver Cancer (BCLC) stage, higher male prevalence and more liver cirrhosis as well as elevated alpha-fetoprotein (AFP), carbohydrate antigen 19–9 (CA19-9), eosinophil, complement 3 (C3), and complement 4 (C4) values. C. sinensis infection correlated with shorter overall survival (OS) (p < 0.05) and recurrence-free survival (RFS) (p < 0.05). Furthermore, Cox multivariate analysis revealed that C. sinensis infection was an independent prognostic factor for OS in HCC patients. Importantly, C. sinensis infection upregulated the expression of HCC cancer stem cell markers CK19 and EpCAM. Conclusion HCC patients with C. sinensis infection exhibit a poor prognosis following hepatectomy. Moreover, C. sinensis infection promotes the acquisition of cancer stem cell-like characteristics, consequently accelerating the malignant progression of HCC. Author summary Clonorchis sinensis (C. sinensis) is a prominent food-borne parasite prevalent in regions such as China, particularly in Guangxi. C. sinensis has been associated with various hepatobiliary system injuries, encompassing inflammation, periductal fibrosis, cholangiocarcinoma and even hepatocellular carcinoma (HCC). A substantial body of evidence links C. sinensis to cholangiocarcinoma, However, the connection between C. sinensis and HCC and the intricate mechanisms underlying its contribution to HCC development remain incompletely elucidated. Our study demonstrates clear clinicopathological associations between C. sinensis and HCC, such as gender, BCLC stage, liver cirrhosis, MVI, AFP, CA19-9, circulating eosinophils and complements. Furthermore, we found that the co-occurrence of C. sinensis exhibited a significant association with shorter OS and RFS in patients diagnosed with HCC. A major finding was that C. sinensis infection promotes the acquisition of cancer stem cell-like characteristics, consequently accelerating the malignant progression of HCC. Our results provide a more comprehensive comprehension of the interplay between C. sinensis and HCC, shedding fresh light on the carcinogenic potential of C. sinensis.


Introduction
Hepatocellular carcinoma (HCC) remains an important global health burden, contributing substantially to cancer-related mortality worldwide [1,2].Notably, China shoulders the highest burden of HCC in the world.In 2019, approximately 300,000 people in China were diagnosed with HCC, which tragically resulted in nearly 200,000 HCC-related deaths [3].Despite recent advancements, the prognosis for HCC patients remains grim, with an overall 5-year survival rate of 25-30% and recurrence/metastasis rates of 50-70% within 5 years following radical resection [3].Chronic viral hepatitis, alcohol-associated cirrhosis, and nonalcoholic fatty liver disease have been proven to be the main risk factors for developing HCC [1,[4][5][6][7][8].Mounting evidence suggests that certain parasitic infections, such as Clonorchis sinensis (C.sinensis), may play a role in the development and prognosis of HCC [9][10][11].
C. sinensis, recognized as one of the most severe food-borne parasites, predominantly infects the biliary system of humans and other mammals [12].Its prevalence is notably high in East Asian regions, including China, Korea, and Vietnam, presenting an important public health concern [13,14].Notably, China bears the greatest burden of C. sinensis infection, with an estimated 35 million people infected worldwide, of which approximately 15 million cases are in China [14,15].The presence of C. sinensis in the bile ducts can lead to biliary inflammation, biliary obstruction, liver cirrhosis and even carcinogenesis [10,[16][17][18].Furthermore, epidemiological and clinical studies have unveiled disconcerting links between C. sinensis infection and the prognosis of HCC patients, suggesting that it may serve as a substantial risk factor for HCC [10].The carcinogenesis of C. sinensis infection involves various factors, such as mechanical obstruction and damage to the biliary barrier system, consisting of infectionrelated inflammation, pathological effects from excretory-secretory products (ESPs), enhanced features of epithelial-mesenchymal transition, and immunopathology imbalance [10,[19][20][21].However, the intricate mechanisms underlying C. sinensis's contribution to HCC development remain incompletely elucidated.
Cancer stem cells (CSCs) account for a small subset of tumor cells with long-term tumorigenic capacity and play a pivotal role in cancer development and therapy resistance [22].Emerging evidence has underscored that the stemness of liver cancer cells is a pivotal contributor to the recurrence and metastasis of HCC [23].Tumors that harbor an abundant CSC population may signal a poor clinical outcome in patients with HCC [24].The CSC biomarkers of HCC include epithelial cell adhesion molecule (EpCAM), CK19, CD44, CD133, CD24, and CD13 [24].Further investigations are needed to explore whether C. sinensis promotes the stemness of liver cancer cells.
Hence, systematic endeavors have been undertaken to scrutinize the correlation between C. sinensis infection and clinical parameters in HCC, aiming to explore the predictive significance of C. sinensis infection in patients with HCC following hepatectomy.Furthermore, this article specifically focuses on the involvement of C. sinensis in liver cancer stemness, with the goal of uncovering the underlying mechanisms responsible for the unfavorable prognosis observed in HCC patients concomitantly afflicted by C. sinensis.

Ethics statement
This research was approved by the Medical Ethics Committee of Guangxi Medical University Cancer Hospital (LW2023135) and conducted following the ethical principles outlined in the Helsinki Declaration of 1964 and its subsequent amendments or other ethical standards with equivalent requirements.To ensure patient confidentiality, the identities of the individuals included in this study were anonymized using computer-generated ID numbers.On admission, all patients provided written consent for their anonymized medical data to be analyzed and published for research purposes.

Study population and data collection
This study was conducted at Guangxi Medical University Cancer Hospital from October 2013 to December 2021.This study conformed to the ethics of the Declaration of Helsinki and was approved by the Ethics Committee of Guangxi Medical University Cancer Hospital.A total of 1,908 HCC patients without cardiovascular diseases (including hypertension and coronary heart disease), chronic respiratory diseases and diabetes mellitus underwent radical resection.The inclusion criteria were as follows: (1) histologically confirmed diagnosis of HCC through postoperative pathological analysis; (2) the absence of prior anticancer treatment; (3) the absence of concurrent malignancies; (4) availability of comprehensive laboratory, pathological, and follow-up data.Exclusion criteria, on the other hand, included the following: (1) previous antitumor treatments such as transcatheter arterial chemoembolization, chemotherapy, or radiofrequency ablation (N = 187); (2) presence of malignancies other than HCC (N = 72); (3) cases of recurrent HCC (N = 143); and (4) unavailability of comprehensive laboratory, pathological, and follow-up data (N = 667).
Clonorchiasis is caused by infection with C. sinensis [25], and the diagnostic criteria for clonorchiasis are as follows, with any one of the subsequent conditions deemed sufficient for establishing a diagnosis [9,12,18,26,27]: (1) preoperative imaging (MRI, CT, microscopy or ultrasound) confirming the presence of C. sinensis eggs or adult worms on the intrahepatic bile ducts; (2) a preoperative positive result in serologic ELISA testing; (3) intraoperative or postoperative pathological examination revealing the presence of adult C. sinensis in the liver or gallbladder; (4) preoperative fecal examination showing the presence of C. sinensis eggs.Finally, according to whether HCC patients were infected with C. sinensis, we assigned them to a C. sinensis (+) HCC group (C.sinensis positive) or a C. sinensis (-) HCC group (C.sinensis negative).Consequently, the clinicopathological data related to HCC following hepatectomy retrospectively analyzed in this study comprised that of 839 patients, among whom 87 exhibited concomitant C. sinensis infection along with HCC.

Laboratory methods
The concentrations of TBiL, ALT, AST, IgG, IgM, IgA, C3 and C4 were determined utilizing a Siemens ADVIA 2400 chemistry analyzer.Hematological parameters were assessed through a Mindray Coulter CAL8000 blood analyzer.Meanwhile, the levels of AFP, CA19-9, and CEA were quantified by employing an Abbott I2000SR analyzer.The proportions of circulating CD4 T lymphocytes, CD8 T lymphocytes, B lymphocytes, and NK cells were analyzed utilizing BD flow cytometry.

Follow-up routine
Patient follow-up was diligently managed by professional staff, utilizing a telephone contact or outpatient monitoring systems to ascertain patient disease status or date of mortality.The identification of tumor recurrence hinged on an analysis of radiological observations extracted from CT or MRI scans, with a particular emphasis on discerning characteristic enhancement patterns indicative of intrahepatic recurrence.When dealing with extrahepatic tumors or those displaying atypical HCC imaging traits, verification was secured through biopsy.Patients underwent systematic monitoring at designated time intervals after their surgical procedures.The calculation of overall survival (OS) entailed determining the span between the date of hepatectomy and the date of death or the last follow-up, with September 30, 2022, as the end date.Recurrence-free survival (RFS) was defined as the temporal gap between the date of hepatectomy and the occurrence of HCC recurrence or the last follow-up, again until September 30, 2022.

Histology and immunohistochemistry
Paraffin-embedded tissue samples from HCC patients were skillfully sectioned into slices measuring 4 μm in thickness.These sections underwent deparaffinization using xylene and were subsequently rehydrated through a series of alcohol washes.To ensure optimal quality, all sections underwent microwave-based heating for repair, while endogenous peroxidase activity was quenched using a 3% H 2 O 2 solution.Following these preparatory steps, the sections were subjected to an overnight incubation at a temperature of 4˚C utilizing anti-CK19 antibody (dilution 1:150; Maixim, Kit-0030) or anti-EpCAM antibody (dilution 1:500; Thermo Fisher, Clone: 1B7).The subsequent immunohistochemical analysis was executed using the DAKO EnVision detection system.
To ensure unbiased evaluation, two independent pathologists, who remained unaware of the associated clinical data, meticulously assessed the immunohistochemical staining scores for CK19 and EpCAM within the liver tissues by lab standard operation files.This evaluation was conducted through a semi-quantitative approach.This evaluation was conducted through a semiquantitative approach.The staining scores were categorized into four distinct levels as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong).Specifically, high expression was defined as a staining score exceeding 2, wherein a minimum of 75% of malignant cells exhibited positive staining.In contrast, moderate expression referred to a staining score of 2, accompanied by at least 25% of malignant cells showing positive staining.Last, low expression entailed a staining score below 2, denoting that less than 25% of malignant cells exhibited positive staining.

Statistical analysis
Statistical analysis was conducted using IBM SPSS Statistics software version 26.0 and R version 4.2.1.Intergroup differences for categorical data presented as ratios were compared using either the Chi-square test or Fisher's exact test.The Mann-Whitney U test was used to compare nonnormal continuous data.Survival curves were generated using the Kaplan-Meier method, and intergroup comparisons of OS and RFS rates were performed using the log-rank test.Univariate and multivariate prognostic analyses were conducted using the Cox regression model.In the multivariate Cox analysis, variables that demonstrated statistically significant differences (p < 0.05) in the univariate analysis were included to identify independent risk factors for OS and RFS.All statistical tests were two-sided, and p < 0.05 was considered statistically significant.

The association between C. sinensis infection and poor prognosis in HCC patients
After a median follow-up of 30 months (range 4-105 months), the three-and five-year OS rates in the whole series were 66.1% and 56.6%, respectively.At the last follow-up, 548 (65.3%)  2 and 3), C. sinensis positivity was an independent prognostic risk factor for OS (hazard ratio = 1.528, confidence interval 95% 1.067-2.187,p < 0.05).However, C. sinensis was not an independent prognostic factor for RFS.

C. sinensis promotes HCC by activating HCC stemness
We hypothesized that C. sinensis promotes HCC by activating HCC stemness.Positive reactivity for CK19 was observed in 30.C. sinensis is a prominent food-borne parasite prevalent in regions such as China, particularly in Guangxi, Guangdong, Heilongjiang and Liaoning [12,29].This parasite has been associated with various hepatobiliary system injuries, encompassing inflammation, periductal fibrosis, cholangiocarcinoma, and even HCC [20,30].While a substantial body of evidence links clonorchiasis to cholangiocarcinoma [18,31,32], the connection between clonorchiasis and HCC is less acknowledged.In alignment with Yuan Kuan Li et al.'s research on the correlation between C. sinensis infection and HCC [9], our results revealed that C. sinensis was highly related to HCC malignant behavior as exemplified by the BCLC stage and MVI.It was believed that the disparity in C. sinensis infection rates between sexes primarily stemmed from dietary habits, particularly the consumption of raw fish [33].This behavior has contributed to a higher prevalence of C. sinensis infection among males compared to females [9,29].In the Guangxi Zhuang Autonomous Region, 16.44% of HCC patients were statistically found to be infected with C. sinensis.Additionally, the prevalence of C. sinensis in males (14.0%, 95% CI, 13.3-14.8)was significantly higher than in females (7.2%, 95% CI, 6.7-7.8),aligning with the elevated incidence of HCC in males compared to females within the Chinese population.Given these findings, a correlation between the phenomena of C. sinensis infection and HCC carcinogenesis is evident.However, this correlation does not translate to a strong causation in the absence of experimental validation.Therefore, further experimental validation is necessary to clarify the impact of C. sinensis infection on HCC carcinogenesis [34][35][36].Our data also indicated a higher prevalence of males in the C. sinensis (+) HCC group than in the C. sinensis (-) HCC group.It has been widely recognized that C. sinensis and other parasitic infections can lead to elevated circulating eosinophils [37].Moreover, our data also showed circulating eosinophils were more elevated in the C. sinensis (+) HCC group than in the C. sinensis (-) HCC group.Eosinophils exert a range of biological effects against helminth parasites, leading to inflammation and damage to affected tissues and even cancers [38,39].Notable studies have also linked estrogen's regulatory role to modulating eosinophil values and function [40].It would be intriguing to explore whether there is a sex-based component to the elevation of eosinophil levels in C. sinensis-infected HCC.To further explore the underlying mechanisms of eosinophil elevation in C. sinensis-related HCC, the roles of sex hormones are also worth investigating.
Importantly, with this study, we are the first to report that C. sinensis (+) HCC patients exhibit elevated circulating complement 3 and 4 levels compared to their C. sinensis (-) HCC counterparts.Complement 3 and 4 have previously shown high predictive accuracy in distinguishing HCC from controls with chronic HCV infection [41].Both C3 and C4 have emerged as constituents of tumor-promoting inflammation, implicated in immune modulation by activating protumorigenic neutrophils and tumor-associated macrophages as well as inducing PI3K/AKT-dependent tumor cell proliferation and epithelial-mesenchymal transition [42,43].However, scant attention has been given to the role of C. sinensis-mediated Complement 3 and 4 in HCC malignant behavior.Further elucidation is warranted to clarify the impact of C. sinensis infection on the development of HCC through Complement 3 and 4 mediation.Cancer stem cells orchestrate a spectrum of crucial roles in hepatocellular carcinoma growth, invasion, metastasis, recurrence, and therapy resistance [44].C. sinensis, a carcinogenic human liver fluke, fosters chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even carcinogenesis through prolonged infection [17].Recent research highlights various mechanisms through which C. sinensis contributes to HCC progression.The excretory/secretory products of C. sinensis inhibit HCC cell apoptosis via mitochondria-mediated pathways triggered by Ca 2+ disruptions [11,30].Additionally, C. sinensis granule protein promotes liver cell malignant transformation, migration, and invasion by activating the ERK and PI3K/AKT pathways [10].However, the role of C. sinensis in promoting human HCC development via cancer stemness regulation remains less understood.
Our findings underscore that C. sinensis not only serves as an independent risk factor for OS and RFS but also elevates the expression of HCC CSC markers, namely, CK19 and EpCAM.CK19+ HCC is an aggressive subtype characterized by its propensity for early recurrence, metastasis and chemotherapy tolerance as well as poor prognosis [45][46][47][48].Oriana Miltiadous et al. reported an independent association between CK19 and HCC recurrence, with a hazard ratio of 2.95 [49].Meanwhile, the EpCAM+ HCC subtype exhibited characteristics of hepatic stem cells, displaying the capacities for self-renewal and differentiation, as well as the capability to initiate highly invasive HCC [50][51][52].This leads us to propose an additional mechanism by which C. sinensis infection promotes HCC progression that involves the enhancement of cancer stemness in tandem with the above three mechanisms.
Mounting evidence indicates that C. sinensis infection drives Th2 immune responses and facilitates the production of the cytokines IL-13, TGF-β, IL-10 and IL-4 [53,54], which have been implicated in promoting cancer stemness [55,56].Carlo De Salvo et al. uncovered the importance of eosinophils in the early cascade leading to intestinalized metaplasia in gastritisprone mice, suggesting a potential mechanism that contributes to the inflammation-metaplasia-dysplasia-carcinoma sequence [57].Our observations revealed higher eosinophil counts in C. sinensis (+) HCC patients than in C. sinensis (-) HCC patients.The underlying mechanism through which C. sinensis infection drives eosinophils or Th2 immune responses affecting HCC stemness needs to be fully investigated.While our study has yielded promising outcomes, there are still some limitations to be considered.First, the sample size, particularly within the cohort of C. sinensis-related HCC patients, is relatively modest.To validate and reinforce our findings, a more extensive sample size is essential.Second, our investigation primarily centered around the correlation between C. sinensis infection and circulating immune cells, neglecting potential alterations within the HCC tumor microenvironment.Expanding our focus to include these changes could provide a more comprehensive understanding.Third, although our study has illuminated that C. sinensis contributes to heightened HCC stemness, the precise underlying mechanism behind this phenomenon remains unexplored.Investigating this mechanism is imperative to fully grasping the intricacies of our findings.
Our research uncovers a hitherto overlooked association between C. sinensis infection and HCC, offering crucial insights into both clinical implications and underlying mechanisms.This study underscores the importance of incorporating C. sinensis infection into the assessment and management of HCC patients, particularly in regions where this parasitic infection is endemic.Given the observed diminished prognosis among HCC patients with C. sinensis infection following hepatectomy, treatment strategies may necessitate targeted interventions tailored to address this specific subgroup.Public health policies may benefit from the integration of systematic screening for C. sinensis in populations at heightened risk by enhancement of early detection and intervention.Future research endeavors should delve into the molecular mechanisms through which C. sinensis infection influences the initiation and progression of HCC.A comprehensive understanding of these pathways holds the potential to inform targeted treatment and intervention strategies.
In conclusion, our study provides robust evidence that C. sinensis infection is strongly linked to unfavorable prognoses in HCC patients due to its capacity to enhance HCC stemness.These results contribute to a more comprehensive comprehension of the interplay between C. sinensis and HCC, shedding fresh light on the carcinogenic potential of C. sinensis.To solidify these findings, further endeavors should encompass large multicenter randomized controlled studies and comprehensive investigations into the intricate mechanisms at play.
38% of C. sinensis (+) HCC and 20.09% of C. sinensis (-) HCC lesions.Positive reactivity for EpCAM was observed in 40.00% of C. sinensis (+) HCC and 15.71% of C. sinensis (-) HCC lesions.The results showed that the expression levels of CK19 (p < 0.05, Fig 3A) and EpCAM (p < 0.05, Fig 3B) in liver cancer tissues of C. sinensis-infected HCC patients were significantly higher than those in noninfected C. sinensis patients.Fig 3C and 3D are representative images of the expression levels of CK19 and EpCAM in C. sinensis (-) HCC and C. sinensis (+) HCC, respectively.

Table 1 . Patient demographics and clinical characteristics.
Fig 2B).A total of 31 different factors were included in the multivariate Cox regression analysis for OS and RFS.Univariate analysis was used to analyze factors that could influence OS or RFS in HCC patients, including C. sinensis.In this study, 15 variables were identified as important predictive factors for OS, including C. sinensis.In the multivariate analysis (Tables patients were alive, including 144 (17.2%) without evidence of disease.In the C. sinensis (-) HCC group, a total of 274 (36.4%) cases of death were recorded, whereas in the C. sinensis (+) HCC group, 47 (54.0%) cases of death were documented.C. sinensis (+) HCC patients had worse OS than C. sinensis (-) HCC patients (at three years, 67.8% vs. 52.4%;atfiveyears, 58.6% vs. 41.5%,p<0.05,Fig2A).The enrolled patients had an RFS follow-up period of 2 to 101 months, with a median of 17 months.RFS rates were lower in C. sinensis (+) HCC patients than in C. sinensis (-) HCC patients (at three years, 34.9% vs. 25.1%; at five years; 18.6% vs. 12.6%, p<0.05,